Post on 26-Dec-2019
1
In Vivo Blunt-End Cloning Through
CRISPR/Cas9-Facilitated Non-Homologous
End-Joining
Jonathan M. Geisinger, Sören Turan, Sophia Hernandez, Laura P. Spector,
Michele P. Calos*
Department of Genetics, Stanford University School of Medicine, Stanford, California, United
States
*For correspondence: calos@stanford.edu
Abstract
The ability to precisely modify the genome in a site-specific manner is extremely useful.
The CRISPR/Cas9 system facilitates precise modifications by generating RNA-guided double-
strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with
a high level of precision by non-homologous end-joining in mammalian cells. We present a
method called knock-in blunt ligation for exploiting this excision and repair to insert
exogenous sequences in a homology-independent manner without loss of additional
nucleotides. We successfully utilize this method in a human immortalized cell line and induced
pluripotent stem cells to insert fluorescent protein cassettes into various loci, with efficiencies
up to 35.8% in HEK293 cells. We also present a version of Cas9 fused to the FKBP12-L106P
destabilization domain for investigating repair dynamics of Cas9-induced double-strand
breaks. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant
increase the repertoire of precision genome engineering approaches.
Introduction
The ability to make precise double-strand
breaks (DSBs) in the genome is extremely useful for
genome engineering, as this ability can facilitate
directed changes in the genome for
applications ranging from studying a gene to
engineering an entire biosynthetic pathway. The most
popular tools for making DBSs are currently zinc
finger nucleases (ZFNs), transcription activator-like
effector nucleases (TALENs), and the clustered
regularly interspaced short palindromic repeat
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(CRISPR)/Cas9 system. Of these three, the
CRISPR/Cas9 system offers the greatest ease of use.
The CRISPR/Cas9 system, a component of the
bacterial RNA-mediated adaptive immune system,
consists of transcribed guide-RNAs derived from
integrated fragments of phage and plasmid DNA that
direct the Cas9 RNA-guided DNA endonuclease to
targets containing complementary sequences to the
CRISPR (Barrangou et al., 2007; Jinek et al., 2012).
The complex requires a protospacer adjacent motif
(PAM) downstream of the target sequence to begin
binding (Sternberg, Redding et al., 2014). Upon
binding, Cas9 generates a blunt DSB three to four
bases upstream of the PAM through the use of RuvC-
like and HNH nuclease domains (Jinek et al., 2012).
Recently, the CRISPR/Cas9 system from
Streptococcus pyogenes has been adapted for genome
editing (Jinek et al., 2012; Mali, Yang et al., 2013;
Jinek et al., 2013; Cong et al., 2013), and has been
successfully used in many eukaryotes (Friedland et al.,
2013; Kim, Ishidate et al., 2014; Zhang, Koolhass, and
Schnorrer, 2014; Gratz, Ukken et al., 2014; DiCarlo et
al., 2013; Harel et al., 2015), as well as in disease and
therapeutic modeling (Xue, Chen, Yin et al., 2014;
Heckl et al., 2014; Platt, Chen et al., 2014; Ousterout
et al., 2015; Hu, Kaminski et al., 2014; Wang and
Quake, 2014). However, the spectre of off-target
cleavage may present significant challenges to the use
of CRISPR/Cas9 in these applications. The chimeric
single-guide RNA (sgRNA) not only tolerates
mismatches, insertions, and deletions throughout its
target sequence, particularly outside of the seed
sequence, but may also display cell type-specific off-
target effects (Fu et al., 2013; Hsu et al., 2013;
Pattanayak et al., 2013; Cho et al., 2014; Lin et al.,
2014; Fu et al., 2014; Wu et al., 2014; Kuscu, Arslan et
al., 2014; Cencic, Miura et al., 2014). Contrary to
these observations, off-target cleavage in murine
embryos and human induced pluripotent and
embryonic stem cells (hiPSCs and ESCs) has been
reported to be extremely rare (Yang, Wang et al., 2013;
Veres et al., 2013; Xie et al., 2014; Yang, Grishin,
Wang et al., 2014).
This discrepancy may be explained by the
underlying repair mechanism of CRISPR/Cas9-
induced DSBs. Typically, these DSBs are thought to be
repaired by error-prone NHEJ, similarly to those
generated by the FokI domains of TALENs and ZFNs
(Jinek et al., 2013; Mali, Yang et al., 2013; Cong et al.,
2013). However, Cas9 makes blunt DSBs, whereas
FokI dimers make overhangs. Thus, these blunt,
chemically unmodified DSBs could be repaired by the
precise NHEJ pathway, which has been previously
demonstrated using the Tn5 transposon system, which
also makes blunt DSBs (van Heemst et al., 2004).
Indeed, precise NHEJ has been observed to resolve the
majority of CRISPR/Cas9-induced translocation
events in human immortalized cells (Ghezraoui et al.,
2014). Additionally, the predominant use of precise
NHEJ may explain the relatively low level of error-
prone editing observed in hiPSCs (Mali, Yang et al.,
2013).
There appears to be a large amount of
variation in sgRNA efficiency, as measured by error-
prone repair between different cell types (Mali, Yang et
al., 2013). Because Cas9-mediated cleavage generates
DSBs with blunt ends, we hypothesized that the error-
prone NHEJ is a by-product of the propensity of Cas9
to remain bound to its target DNA sequence after
cleavage (Sternberg, Redding et al., 2014). Thus,
precise NHEJ should be the contributing factor to
resolving a significant percentage of Cas9-mediated
DSBs, potentially explaining the large degree in
variation. We further hypothesized that precise NHEJ
could be exploited to increase the specificity of Cas9-
based genome editing, which has also been suggested
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based on experiments in zebrafish (Auer et al., 2014).
Additionally, we sought to restrict the window of Cas9
activity to limit both the amount of potential off-target
activity and DNA-damage-associated cell cycle delay,
which has been demonstrated in budding yeast
(Demeter et al., 2000).
Here, we demonstrate that, by generating two
DSBs with two different sgRNAs at the same time,
both large and small precise deletions can be
generated, that these DSBs can be exploited to knock
in various expression cassettes and gene modifications
through precise NHEJ, and that this strategy is
applicable in both human and mouse cells.
Additionally, we present a destabilized variant of Cas9,
which we originally designed to facilitate greater
control over genome modification, but which was
repurposed for investigation of the repair dynamics
and outcomes of Cas9-mediated DSBs.
Results
Patterns of Excision and Repair Resulting
from Paired PAM Orientations
Because Cas9 generates blunt DSBs, we
hypothesized that these blunt ends would be
preferentially repaired by precise NHEJ. To test this
hypothesis, we chose to generate two DSBs within the
same locus rather than one because precise NHEJ
mediated by one individual sgRNA would be
indistinguishable from uncleaved DNA at the sequence
level. However, if a cell received two sgRNA vectors
expressing two different sgRNAs, there would be a
high probability of excision of the intervening
sequence between the paired sgRNA targets, allowing
for religation of the genomic junctions and subsequent
detection and analysis via PCR and Sanger sequencing
(Figure 1A). Others (Cong et al., 2013; Mali, Yang, et
al., 2013; Canver, Bauer et al., 2014) have also
observed this resulting excision and religation. This
excision may occur because Cas9 appears to bind the
PAM side of the DSB less strongly after cleavage
(Sternberg, Redding et al., 2014). A high level of
precision has also been observed in translocations
generated by two sgRNAs (Ghezraoui et al., 2014).
However, the repair consequences of paired PAM
orientations resulting from using two sgRNAs
simultaneously have not been explored. For pairs of
sgRNAs, there are four possible orientations: 1) PAMs
Out, 2) PAMs In, 3) PAMs 5’, and 4) PAMs 3’ (Figure
1B). It is important to note that PAMs 5’ and PAMs 3’
are functionally equivalent at first glance. Because
Cas9 can cleave at either the third or fourth base
upstream of the PAM, precise rejoining can occur with
0, +1, or -1 bases on each of the repaired genomic
ends, depending on which PAM orientation is used. As
such, each orientation has four possible outcomes for
precise repair (detailed in Figure 1B and shown as
5’sgRNA bases/ 3’ sgRNA bases). To this end, we
generated multiple sgRNAs against the 3’UTRs of
human MYOD1, PAX3, PAX7, and mouse Utrophin
(Utrn) to examine the repair patterns and degree of
precision utilized by paired PAM orientations (Figure
1C-E).
To date, the largest study of repair patterns of
paired PAM-mediated excisions focused only on the
PAMs 3’ orientation (Canver, Bauer et al., 2014). In
our study, we first sought to examine if there were any
large differences in repair patterns between all four
orientations. Each pair was tranfected separately into
HEK293 cells. In examining all four paired PAM
orientations at the MYOD1 3’UTR in HEK293 cells, we
observed a remarkably high level of precision of NHEJ
repair as indicated by the high frequency (64.71-
96.15% of observed amplicons) of precise repair,
regardless of orientation (Figure 1Ci and ii; for
sequences, see Figure 1-figure supplement 1). PAMs 5’
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showed the highest frequency of precise repair,
reaching 96.15%, whereas PAMs 3’ showed the lowest
frequency of precise repair (Figure 1Cii). Imprecise
repair was mainly limited to small deletions and
occasional inclusion of bases that should have been
excised. PAMs Out and PAMs 3’ displayed a deviation
from 0/0 precise repair, with both possessing a mostly
0/+1 precise repair pattern, which may be the result of
them sharing the same 3’ sgRNA (Figure 1Ciii).
Interestingly, the predominance of 0/+1 precise repair
observed with PAMs 3’ appears to be due to 0/0 repair
generating a perfect target site for the 5’ sgRNA in this
pair. Comparing the deviations from 0/0 precise
repair between PAM orientations revealed that there
was a significant difference between distributions (P <
0.0001; Kruskal-Wallis test). Post hoc multiple
comparisons revealed no significant difference
between PAMs Out and PAMs 3’ or between PAMs In
and PAMs 5’ (P > 0.9999 for both comparisons), but
that all other comparisons between orientations were
significantly different (P < 0.0001; Dunn’s multiple
comparisons test for all comparisons). Additionally,
we observed only one stereotypical error-prone event
(Figure 1-figure supplement 1). These findings suggest
that there are no inherent differences in repair
precision between paired PAM orientations, the
differences are dependent on individual sgRNAs, and
NHEJ repair of CRISPR/Cas9-induced DSBs is not
inherently error-prone.
Having observed that all four orientations
were capable of facilitating precise repair at one locus,
we next asked if different paired PAMs in the same
orientation would display the same degree of repair
precision at the same locus. Thus, we utilized four
pairs of sgRNAs in the PAMs In orientation at the
PAX3 3’UTR in HEK293 cells (Figure 1Di). We
observed that the vast majority of sequenced deletion-
containing amplicons for all four pairs were
remarkably precise, with the frequency of precise
repair ranging from 87.5% to 100% (Figure 1Dii;
Figure 1-figure supplement 2). Interestingly, we did
not observe insertions in any of these sequenced
amplicons. We observed that there was a significant
difference between the distributions of deviations (P <
0.0001; Kruskal-Wallis test), but that only PAMs In #1
displayed a noticeable deviation from 0/0 precise
repair. The majority of these sequenced amplicons
were of the 0/-1 precise repair variety, which was
found to be significantly different than that of the
other three pairs (P < 0.004 for PAMs In #1 versus
each other pair; post hoc Dunn’s multiple comparisons
test for all comparisons; Figure 1Diii). These findings
provide additional evidence for both the high degree of
precision involved in CRISPR/Cas9-induced NHEJ
repair, and that differences in repair are dependent on
individual sgRNAs, possibly based on target sequence.
Having observed that CRISPR/Cas9-induced
NHEJ repair does not appear to be inherently error-
prone, we next sought to examine whether a given pair
of sgRNAs generates similar repair patterns in
different cell types. Thus, we designed pairs of
sgRNAs against the PAX7 3’UTR, which were
subsequently transfected into HEK293 cells and H9
human ESCs (hESCs) (Figure 1Ei). We observed a
mostly high level of precise repair in the H9 hESCs,
with only one pair resulting in less than 75% precise
repair (Figure 1Eii; Figure 1-figure supplement 3 for
HEK293; Figure 1-figure supplement 4 for H9 hESCs).
However, we observed what appeared to be a more
varied level of precise repair in HEK293 cells with the
same PAM pairs, ranging from 44.44% to 90.48%
(Figure 1Eii). Additionally, we only observed two
typical error-prone events for HEK293, one resulting
from the use of PAX7r1-4 and a double event for
PAX7r1-4 and PAX7r1-1 (Figure 1-figure supplement
3). For H9 hESCs, we observed two typical error-
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prone repair events: one for PAX7r1-2 and one for
PAX7r1-3 (Figure 1-figure supplement 4). In spite of
this variation, deviation from 0/0 precise repair was
not significant for any given pair between cell types. In
fact, for each pair, the deviation from 0/0 precise
repair was not significantly different between cell types
(P > 0.8 for all four comparisons; Dunn’s multiple
comparisons test; Figure 1Eiii). These data illustrate
that a given pair of sgRNAs generates similar patterns
of repair with similar degrees of precision in different
cell types of the same species.
Given that we observed a high degree of
precise repair in human cells across three loci and 16
pairs of sgRNAs, we sought to examine if a high level
of repair precision would also be observed in mouse
cells. Paired sgRNAs have been previously utilized to
generate deletions in murine embryos, although there
was significant variability in deletion size between
embryos (Zhou, Wang et al., 2014). To examine
repair patterns in mouse cells, we transfected C2C12
murine immortalized myoblast cells with three pairs of
sgRNAs in different orientations against the mUtrn
3’UTR (Figure 1Fi). We observed not only a more
varied pattern of repair than with human cells (precise
repair ranged from 41.67% to 100% of amplicons;
Figure 1Fii; Figure 1-Figure supplement 5), but also a
higher degree of repetition in the imprecise excision
repair amplicons in their deviation from 0/0 precise
repair (Figure 1Fiii). Interestingly, we found that there
was no significant difference between the distributions
of deviance from 0/0 precise repair for the three
orientations (P = 0.1354; Kruskal-Wallis test). These
results indicated that CRISPR/Cas9 facilitates a high
level of repair in murine cells as well, but may
potentially have generated a more varied distribution
of imprecise repair due to species-specific differences
in NHEJ repair.
Knock-in Blunt Ligation: CRISPR/Cas9-
Mediated in vivo Blunt-End Cloning
Because of the high level of precise NHEJ
repair we observed, we asked if we could exploit this
apparent in vivo blunt-end ligation to replace
endogenous sequences precisely with linear exogenous
sequences in a homology-independent manner using
CRISPR/Cas9 (Figure 2A). A similar method has been
developed in zebrafish using plasmid DNA in which
the same sgRNA is used to cleave both the genome and
the plasmid, ultimately resulting in homology-
independent knock-in of the plasmid, albeit with
reduced levels of perfect ligation (Auer et al., 2014).
To test this idea and examine the level of repair
precision, we constructed an expression cassette
consisting of the mouse PGK promoter driving
expression of the PuroRΔTK fusion gene linked via a
P2A skipping peptide to mCherry, followed by the
rabbit beta globin terminator sequence (PTKmChR).
We also constructed another cassette containing the
same sequences, but flanked by a phiC31 attP site
upstream of the PGK promoter and a Bxb1 attP site
downstream of the terminator sequence for the initial
purpose of generating a selectable landing pad that did
not rely on homology arms for integration (DICE-
EPv2; Figure 2B). Each cassette was amplified via
PCR with 5’ phosphorylated primers, purified, and
subsequently co-transfected into HEK293 cells with
various sgRNA/Cas9 constructs, singly or in pairs,
before being subjected to flow cytometric analysis and
junction PCR, followed by Sanger sequencing (Figure
2C). In this set of experiments, we utilized sgRNAs
targeting the PAX7 3’UTR (Figure 2D). For the DICE-
EPv2 cassette, flow cytometric analysis revealed that
the percentage of mCherry+ cells was increased above
cassette alone (0.20 ± 0.15%) for sgRNAs PAX7r2-1
and PAX7r3-2, both alone and paired (referred to as
PAMs Out Large) with a range 1.61-2.67% two days
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post-transfection, and that the percentage of
mCherry+ cells could be further increased to 17.80-
24.50% with four days of puromycin selection , albeit
at the risk of increasing selection for random
integration (Figure 2E-F; 14.30 ± 0.20% for cassette
alone). For the PTKmChR cassette after two days, we
also observed that the percentage of mCherry+ cells
was increased above cassette alone (0.56 ± 0.32%) in
the presence of all sgRNAs tested, which consisted of
two pairs of PAMs 5’, two pairs of PAMs Out (the four
pairs previously characterized in Figure 1F), and the
PAMs Out Large pair, together and singly for a range
of 2.63-5.74% (Figure 2G). Interestingly, four days of
puromycin selection resulted in a lower percentage of
mCherry+ cells with cassette alone (6.57 ± 1.64%) than
with the DICE-EPv2 cassette. Puromycin selection
also resulted in an increase in the percentage of
mCherry+ cells for the PAMs 5’ and PAMs Out pairs,
ranging from 17.10% to 32.60%.
Subsequent PCR analysis of bulk unsorted
cells for all possible junctions of genomic and cassette
sequences revealed that knock-in blunt ligation of the
DICE-EPv2 cassette led to imprecise repair of the
genomic junction (60% of sequenced amplicons) and
an absence of precise cassette junction repair, whereas
knock-in blunt ligation of PTKmChR cassette resulted
in a high level of precise genomic junction repair
(93.75%) and an appreciable degree of precise cassette
junction repair (37.5%; Figure 2Hi and ii). We
speculated that the lack of precise cassette junction
repair with DICE-EPv2 was attributable to the
formation of hairpin structures by the palindromic
AttP sites. Such hairpins could be unfavorable to
direct end-joining. Thus, these hairpins would be
more subject to removal by nucleases. Additionally,
we observed one instance of an insertion of part of the
pX330 plasmid (similarly to that observed in Canver,
Bauer et al., 2014). These results indicated that the
blunt DSBs generated by the CRISPR/Cas9 system
could be exploited in mammalian cells to knock-in
PCR cassettes in a homology-independent manner.
We observed that cassettes were knocked into the
genome in both orientations, indicating that ligation
by precise NHEJ lacks directionality, as expected due
to lack of homology arms on our cassettes (Figure 2-
figure supplement 1).
Because of the relatively large size of the
DICE-EPv2 and PTKmChR cassettes and the relative
dimness of mCherry, we developed a second series of
smaller, brighter cassettes. These cassettes consist of
the Ef1α promoter driving the expression of a
fluorescent protein, which is followed by the rabbit β-
globin terminator sequence (Figure 3A). Our
fluorescent proteins of choice were Clover, mRuby2
(both from Lam et al., 2012), mKOκ (Tsutsui et al.,
2008), mCardinal (Chu et al., 2014), and mCerulean
(Rizzo et al., 2004) due to minimal spectral overlap
and (for the most part) brightness. These cassettes
were less than 2 kb in size and could be readily
amplified from template plasmid and easily purified.
We called these cassettes the pKER (polymerase-
chain-reaction-based Knock-in EF1α-RBG terminator)
series, and they are depicted by brightness in Figure
3A.
To quickly assess the suitability of the pKER
series for KiBL, we used pKER-mKOκ to investigate
the effects of the orientation of the paired PAMs as
well as the effects of the cassette’s phosphorylation
status on the efficiency of KiBL, using the percentage
of positive cells as a proxy. We reasoned that
unphosphorylated cassettes may have a selective
advantage for KiBL as they may not be detected as
free, broken ends of DNA as readily as phosphorylated
cassettes may be, and they may be decrease the
possibility of amplicon concatemerization. For these
experiments, the sgRNA pairs consisted of all four
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possible PAM orientations directed against the same
region of the human H11 locus on chromosome 22
(Figure 3B), which was previously identified as a safe
harbor site in the human genome (Zhu et al., 2014).
H11 is an intergenic locus between DRG1 and
EIF4ENIF1 on human chromosome 22 (Figure 3-
figure supplement 1). There is a potential non-coding
RNA at this locus, but it has only been observed in
human lung and only from a cDNA screen. We
transfected HEK293 cells with phosphorylated or
unphosphorylated pKER-mKOκ cassettes and pairs of
sgRNA/Cas9 vectors, before subjecting the cells to flow
cytometric analysis two days later (Figure 3C). Two
days post-transfection, we observed a higher
percentage of mKOκ+ cells in the cultures co-
transfected with CRISPR/Cas9 vectors (ranging from
8.15-11.64%) than in those transfected with cassette
alone (1.34-1.49%; Figure 3D). Surprisingly, flow
cytometry did not reveal any significant differences
between PAM orientations or cassette phosphorylation
status in terms of percentage of mKOκ+ cells or
relative normalized median fluorescence intensity
(MFI), other than that the presence of Cas9 increased
both measures (Figure 3E; two-way ANOVA with
repeated measures followed by post-hoc Tukey’s
multiple comparisons test, P < 0.003 for all
comparisons when comparing PAMs-treated to
cassette alone). These results suggested that the pKER
cassettes were well-suited for use in KiBL: positive
cells were bright, and treatment with Cas9 resulted in
a large increase in positive cells.
Stabilized Cas9-DD Combined with Nuclease
Protection Facilitates a Higher Degree of
Precise Repair at the Genome-Cassette
Junction
We and others have observed that, in utilizing
the CRISPR/Cas9 system, successful modifications of
one allele appeared to coincide with undesired
mutations in the sister allele (unpublished data;
Canver, Bauer et al., 2014). The duration of Cas9
activity may be responsible for these additional
modifications. Thus, we initially sought to generate a
more temporally controllable Cas9. We chose to utilize
the FKBP12 L106P destabilization domain because it
was relatively small, has been shown to be effective at
destabilizing a wide range of proteins, and its
stabilizing ligand, the small molecule Shield-1, was
relatively inexpensive and had virtually no effect on
cell viability, as well as good intracellular availability
and potency (Banaszynski et al., 2006; Maynard-
Smith et al., 2007). We initially chose to fuse the
destabilization domain to the C-terminus of SpCas9
because we desired a version of Cas9 that possessed
some residual stability in the absence of Shield-1
(Banaszynski et al., 2006). The resulting fused
domain was in close proximity to the PAM-binding
domain of SpCas9 (Anders et al., 2014). We called this
version of Cas9 “Cas9-DD” (Figure 4A, Figure 4-figure
supplement 1A). Additionally, we also constructed a
version of Cas9 with the destabilization domain fused
to the N-terminus, which we called “DD-Cas9” (Figure
4-figure supplement 1A-B). We then examined the
relative stability of Cas9-DD and DD-Cas9 via Western
blotting of HEK293 cells transfected with constructs
encoding the H11-r1-2 sgRNA and WT-Cas9, Cas9-DD,
or DD-Cas9 (Figure 4-Figure supplement 1C). After 29
hours, we observed a higher level of Cas9-DD protein
present in cells treated with 0.5 µM Shield-1,
compared to cells treated with Cas9-DD vector alone
(destabilized Cas9-DD). Similarly, for DD-Cas9,
treatment with 0.5 µM Shield-1 appeared to increase
the level of DD-Cas9 protein relative to DD-Cas9
vector alone. These results indicated that the addition
of the destabilization domain to Cas9 resulted in a
destabilized protein that could be stabilized by the
addition of Shield-1.
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To characterize how Cas9-DD would affect
KiBL efficiency, we transfected our PAX7 PAMs Out
Large sgRNA pairs on vectors encoding the sgRNAs
and either wild-type Cas9 (WT-Cas9) or Cas9-DD,
along with phosphorylated or unphosphorylated
pKER-Clover cassettes into HEK293 cells, which were
subjected to flow cytometric analysis and harvested for
genomic DNA after two days (Figure 4B). We chose
PAX7 because we had already successfully carried out
KiBL and analyzed the pattern of repair at this locus
(Figure 2). Flow cytometric analysis confirmed that
co-transfection with the sgRNA/Cas9 vectors
increased the percentage of Clover+ cells (18.12-
35.8%) over that of cassette alone (2.36-2.64%; Figure
4C-D; P < 0.002 for all comparisons; Tukey’s multiple
comparisons test). Interestingly, we did not detect any
significant difference between Cas9-DD and WT-Cas9
for the percentage of Clover+ cells or for normalized
MFI (Figure 4D). Additionally, we did not detect any
significant difference between the presence and
absence of 1 μM Shield-1 with Cas9-DD.
Having already observed that WT-Cas9
facilitated knock-in blunt ligation of linear cassettes,
we next sought to determine the effect of Cas9-DD at
the molecular level. To this end, we examined the 5’
PAX7 genomic-5’ pKER-Clover junction resulting from
KiBL using unphosphorylated cassettes with WT-Cas9
or destabilized Cas9-DD with our sgRNAs (Figure 4E,
Figure 4-figure supplement 2). We did not examine
junctions using stabilized Cas9-DD at this time as we
reasoned that its junctions would resemble those of
WT-Cas9. Additionally, we examined whether the
addition of three phosphorothioate bonds in the 5’
ends of the primers used to amplify the cassettes
affected the precision of the cassette side of the
junction by protecting the cassette from nuclease
degradation. We observed that unprotected cassettes
co-transfected with WT-Cas9 resulted in precise
genomic junctions, but imprecise cassette junctions in
100% of observed amplicons (Figure 4F).
Interestingly, protecting the 5’ ends of the cassette only
resulted in a relatively modest increase in precise
genomic/precise cassette junctions (40% of
amplicons) when using WT-Cas9 (Figure 4F). When
destabilized Cas9-DD was used in conjunction with
phosphorothioate bonds, we observed a greater
increase in precise genomic/precise cassette junctions
(60% of amplicons), but, surprisingly, we also
observed a slight increase in imprecise
genomic/imprecise cassette junctions (20% of
amplicons; Figure 4F). These results appeared to
demonstrate that the use of destabilized Cas9-DD,
along with the addition of nuclease protection to the
pKER cassettes, facilitated a higher degree of cassette
junction precision in KiBL.
KiBL results in efficient, precise knock-ins in
human induced pluripotent stem cells
Having observed that the CRISPR/Cas9
system could facilitate the precise knock-in of cassettes
in an immortalized cell line, we sought to apply KiBL
to human iPSCs (hiPSCs). The successful application
of such a method would provide a viable alternative to
traditional nuclease-mediated homologous
recombination (HR). We chose a line of hiPSCs
generated from a healthy donor that we refer to as
JF10, and chose H11 as our target locus utilizing the
PAMs Out sgRNAs. The JF10 cells fortuitously
possessed two allele-specific SNPs, one flanking each
sgRNA target, which allowed us to determine which
allele was targeted for genome editing (Figure 5A). In
order to minimize the effect of untransfected cells on
downstream analyses, we isolated fluorescent-
protein+ cells via FACS four to seven days post-
electroporation with unphosphorylated pKER
cassettes containing phosphorothioate bonds and the
sgRNA/Cas9 vector pairs (Figure 5B). To our surprise,
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9
we observed rather low cell viability and relatively low
numbers of Clover+ cells when using the WT-Cas9
vectors, whereas we observed greater cell viability and
higher numbers of Clover+ cells when using the Cas9-
DD vectors in the absence of Shield-1 (Figure 5C). 0.5
μM and 1 μM of Shield-1 resulted in a moderately
increased level of cell viability compared with WT-
Cas9. FACS analysis revealed that the Clover+ cells
formed a mostly discrete population in these hiPSCs
(Figure 5D). Additionally, the percentage of Clover+
cells was always observed to be less than 0.5% of live
cells (Figure 5E). This low percentage may be due
both to the relatively low amount of transfected
cassette (200ng) and the lower electroporation
efficiency of linear double-stranded DNA. However, we
found that there was a significant difference between
the mean percentages of Clover+ cells (P = 0.0193;
ordinary one-way ANOVA), but that only the mean
percentages of cassette alone (0.069%) and Cas9-DD +
0.5 µM Shield-1 (0.147%) were significantly different
from each other (P = 0.019; post hoc Tukey’s multiple
comparisons test). In order to analyze precise end-
joining at the junctions, we carried out targeted
genomic amplification on pooled Clover+ cells by
amplifying across the H11 locus in order to capture all
of the KiBL events (Figure 5F). Subsequent analysis of
the H11 5’- Clover 5’ junction revealed that, in the
presence of Shield-1, Cas9-DD-induced DSBs resulted
in the precise joining of both the genomic and cassette
sequences, but that the absence of Shield-1 resulted in
the loss of the PAM and the three bases preceding it on
the genomic side, but no loss of bases on the cassette
side (Figure 5G). Additionally, the use of destabilized
Cas9-DD appeared to result in a C-to-T mutation nine
bases 5’ of the PAM. We have only observed this
mutation with Cas9-DD in the absence of Shield-1.
Bulk sequencing of the Clover 3’-H11 3’ junction for
stabilized Cas9-DD also showed precise joining of the
genome with the cassette (Figure 5-figure supplement
1A). When utilizing stabilized DD-Cas9, we observed
precise genomic repair and mostly precise cassette
repair, but we also observed the same genomic
deletion and C-to-T mutation at the 5’ junction as we
had observed with destabilized Cas9-DD (Figure 5-
Figure supplement 1B). When analyzing destabilized
DD-Cas9, we did not observe the presence of 5’ H11-
5’cassette junctions (Figure 5-Figure supplement 2).
These results demonstrated that KiBL is feasible in
hiPSCs and that Cas9-DD may be preferable to WT-
Cas9 due to increased cell viability. Additionally, these
results demonstrated a difference between the
stabilized and destabilized forms of Cas9-DD in that
stabilized Cas9-DD may result in higher precision
KiBL whereas destabilized Cas9-DD may be more
useful for probing repair dynamics of Cas9-induced
DSBs.
We recognized that there was the possibility
that we were repeatedly sampling and sequencing the
same clonal population in our initial hiPSC KiBL
experiments. We attempted to rule out this possibility
in three ways. First, we carried out single-cell PCR
across the H11 locus utilizing FACS to sort single cells
into wells of 96-well plates. From this experiment, we
identified two cells that underwent KiBL: one
originated from treatment with stabilized Cas9-DD
(cell I), the other from destabilized Cas9-DD (cell II)
(Figure 6A). Cell I, interestingly, appeared to have
only received the 5’ sgRNA/Cas9-DD vector and had
precise genomic and cassette junctions for both the 5’-
5’ and 3’-3’ junctions. Sequence analysis revealed that
cell II received both sgRNA vectors and possessed a
precise 5’ genomic junction, but was lacking four bases
of the 5’ cassette junction. Interestingly, the 3’ cassette
junction was precise, but the 3’ genomic junction
appeared to have been cut 16 bases downstream of the
actual PAM site for the 3’ sgRNA (Figure 6A). In both
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10
cells, the other allele appeared be completely
unmodified except for an additional adenosine present
at the cleavage site of the 5’ sgRNA, potentially as the
result of error-prone NHEJ.
To further examine the level of clonality
generated by KiBL, we took advantage of the pKER
cassettes’ limited spectral overlap and co-transfected
1) pKER-Clover and pKER-mKOκ, and 2) pKER-
Clover, -mKOκ, and –mCardinal with our H11 PAMs
Out sgRNA pair into JF10 hiPSCs, which were
subsequently analyzed via FACS. In the first
condition, we observed that Clover+mKOκ- and
Clover-mKOκ+ cells were roughly equivalent in
relative frequency and that Clover+mKOk+ cells made
up a smaller frequency of the population (Figure 6B).
Interestingly, in the second condition, we observed all
three possible single-positive populations, and no
double- or triple-positive populations (Figure 6C).
These data illustrated that there were at least three
clones in each experiment.
To address clonality at the molecular level, we
employed an approach using pKER-Clover cassettes
containing 6-base barcodes and 50-base homology
arms. Homology arms of this size have been
previously used with the CRISPR/Cas9 system to
facilitate homologous recombination in human
immortalized cell lines (Zheng, Cai et al., 2014) and
mouse ESCs (Li et al., 2014). After co-transfection
with the H11 PAMs Out sgRNA pairs and Cas9-DD,
followed by isolation with FACS, sequence analysis of
the 5’ genomic-5’ Clover junction revealed that HR
occurred several times (Figure 6D). After isolation
with FACS, PCR amplification of the 5’ genomic-5’
Clover junction, and subsequent subcloning, we
observed 11 different barcodes in 12 sequenced
amplicons (Figure 6D). Out of these, 11 had a thymine
(the human reference allele, which we had placed in
the 5’ homology arm) instead of cytosine (the allele
possessed by JF10) in the position prior to the PAM
that differed from reference, indicating that
homologous recombination did indeed occur. We also
observed that both alleles underwent HR, as indicated
by detection of both alleles of our allele-specific T/C
SNP on the 5’ side of the locus. Additionally, we
observed one non-barcoded KiBL event and a
barcoded KiBL event where the cassette appeared to
lack at least the 5’ homology arm. We did not observe
any KiBL events where the homology arm was
incorporated via NHEJ rather than HR. These results
indicated that several independent KiBL events
occurred in the Clover+ populations, ruling out
concerns over clonal amplification affecting our
analyses. Additionally, these data demonstrated that
PCR amplicons possessing short homology arms
combined with the CRISPR/Cas9 system facilitated
homologous recombination in hiPSCs, which had not
previously been demonstrated.
Analysis of Off-Target KiBL Events
Because KiBL utilizes the CRISPR/Cas9
system, there was a concern that off-target cleavage
would result in the uptake of cassettes at sites other
than the targeted locus. Indeed, such events have been
observed in HEK293T cells using the GUIDE-seq
method for unbiased identification of CRISPR/Cas9
off-target cleavage events (Tsai et al., 2014b). We
chose to examine the top six off-target sites for each
sgRNA in the H11 PAMs Out pair as determined by the
MIT CRISPR Design tool. For each off-target site, we
attempted to amplify the 5’ genomic-5’ cassette, 3’
genomic-5’ cassette, 5’ genomic-3’ cassette, and
3’genomic-3’ cassette junctions, which led to 48 off-
target reactions per sample. In order to perform such
a large number of assays, we used the multiple
displacement amplification variation of whole genome
amplification to ensure that there would be enough
DNA. We analyzed seven pools of Clover+ cells: one
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11
pool treated with WT-Cas9, two pools treated with
destabilized Cas9-DD, two pools treated with Cas9-DD
and 0.5 μM Shield-1, and two pools treated with Cas9-
DD and 1 μM Shield-1. We were unable to detect off-
target KiBL events at the vast majority of predicted
junctions, with the exception of faint detection of the
3’H11 sgRNA OT4 genomic reverse primer-3’pKER
cassette junction (Figure 7). These data suggest that,
at least in hiPSCs, KiBL occurred predominantly at the
on-target locus. It is also possible that the frequency
of KiBL off-target events fell below the sensitivity of
our assay.
Discussion
We demonstrated in this work that the blunt
cleavage activity of the CRISPR/Cas9 system mediates
repair with a high degree of precision and can be
exploited to precisely insert exogenous sequences into
the genome through knock-in blunt ligation. We
found that all four paired PAM orientations are
capable of facilitating precise repair and that precise
repair can be observed in both human and mouse cells.
We then demonstrated that PCR amplicons lacking
homology arms can be used to precisely replace the
sequence between sgRNA targets and that this in vivo
blunt-end cloning functions in an immortalized cell
line and in hiPSCs. Lastly, we developed a variant of
Cas9 fused to a destabilization domain with the initial
goal of facilitating a greater degree of control over
Cas9 activity.
Other strategies utilizing the CRISPR/Cas9
system to facilitate knock-in of exogenous sequences
into the genome have relied on knocking in plasmid
DNA through NHEJ (Auer et al., 2014) or
microhomology-mediated end-joining (Nakade et al.,
2014). In general, the plasmid-based methods rely on
in vivo cleavage of a plasmid donor to mediate
integration, which was previously demonstrated with
ZFNs (Cristea et al., 2012). These methods introduce
additional undesired sequence into the cell through
the plasmid backbone, whereas KiBL only introduces
the knock-in cassette and the sgRNA/Cas9 vector.
Additionally, the Cas9 nickase has been used to knock-
in a double-stranded oligonucleotide (dsODN) with
overhangs in a strategy similar to the ObLiGaRe
method used with ZFNs, albeit with relatively low
efficiency (Ran, Hsu et al., 2014; Maresca et al., 2013).
Our observation that unphosphorylated cassettes are
capable of ligating into CRISPR/Cas9-induced DSBs at
relatively high frequency in HEK293 cells appears to
contradict observations in the GUIDE-seq method
(Tsai et al., 2014b). However, this finding can be
explained by our much larger cassettes: our
unprotected, unphosphorylated cassettes may be much
less negatively affected by endogenous nucleases than
the 34-bp GUIDE-seq oligos.
Another set of methods for knock-ins has been
developed around exploiting HR and HDR. In
cultured Drosophila cells, the use of PCR amplicons
containing homology arms has been used to great
effect (Böttcher et al., 2014), which is similar to what
has been observed with human immortalized cell lines
and mouse ESCs (Zheng et al., 2014; Li et al., 2014). A
subset of these methods has focused on increasing
HDR frequency through cell cycle synchronization
(Lin, Staahl et al., 2014) and by inhibition of the NHEJ
machinery (Chu et al., 2015; Maruyama et al, 2015).
Our method offers the advantage of functioning during
the G1, S, and G2 phases of the cell cycle, whereas the
HR and HDR strategies are restricted to late S and G2
phases. Thus, our method may be particularly useful
in non-dividing cells, such as neurons where the
application of Cas9 has been limited to generating
mutations through error-prone repair (Swiech,
Heidenreich et al., 2014).
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12
Targeted genome engineering has the
potential to be a useful tool for research, therapeutic,
and industrial applications. The CRISPR/Cas9 system
has the advantages of being relatively inexpensive and
easy to use, but may have larger off-target effects than
TALENs or ZFNs. Several approaches have been put
forward to restrict off-target cleavage, including simply
using less sgRNA and Cas9, using truncated sgRNAs
for increased specificity, using paired nickases,
controlling Cas9 expression through the inducible Tet-
ON system, and splitting Cas9 in half (Hsu et al., 2013;
Fu et al., 2013; Ran, Hsu et al., 2013; Zhu, Gonzalez,
and Huangfu, 2014; Wright, Sternberg et al., 2015;
Zetsche, Volz, and Zhang, 2015). Tet-ON induction,
however, generates a large amount of mRNA, which
could lead to an increase in Cas9 off-target activity.
The split-Cas9 variants, while potentially being more
controllable, have substantially reduced cleavage
activity, relying on either the sgRNA or rapamycin to
bind the two halves together (Wright, Sternberg et al.,
2015; Zetsche, Volz, and Zhang, 2015). Our
destabilized Cas9 retains the simplicity of requiring
only two expression cassettes, rather than three or
four. Additionally, Shield-1 itself does not induce any
undesired responses when applied to cells in culture or
in animals, unlike rapamycin, (Banaszynski et al.,
2006; Maynard-Smith et al., 2007; Banaszynski et al.,
2008). During the preparation of this manuscript, a
fifth variant was developed using an evolved ligand-
dependent intein to restrict the activity of Cas9 until
the binding of the ligand (in the form of 4-
hydroxytamoxifen) results in the cleavage of the intein
and activation of Cas9 (Davis et al., 2015). Such a
system is elegant, but possesses the drawback of being
unable to restrict Cas9’s activity once the intein is
cleaved. It would be interesting to combine the
destabilization domain with this intein because such a
combination could allow for an unprecedented level of
control of Cas9 activity.
Our observation that the use of Cas9-DD in
the absence of Shield-1 leads to the loss of nucleotides
on the genomic side of the genome-cassette junction is
particularly interesting. One possibility is that these
deletions are the result of a transient binding event by
Cas9 as it searches for its programmed target site.
Cas9 has been found to probe potential binding sites
by finding PAMs (Sternberg, Redding et al., 2014), and
unbiased interrogation of Cas9 cleavage sites in
HEK293 cells has revealed that SpCas9 can recognize
non-canonical PAMs (Tsai et al., 2014b). Additionally,
experiments in C. elegans have demonstrated that
choosing sgRNAs that target loci enriched with
potential PAMs increases successful editing (Farboud
and Meyer, 2015), which was motivated by the
observation that Cas9 can be sequestered by
competitor DNA containing a high density of PAM
sequences (Sternberg, Redding et al., 2014). Those
findings, coupled with our observations, suggest that
there may be several low-affinity binding events at
such target sites that can result in cleavage by Cas9. Of
further interest is the co-occurrence of a single C-to-T
transition upstream of the PAM with the nucleotide
loss event. Because neither allele of JF10 possesses a T
at this position and we only observed this mutation
when using destabilized Cas9, we speculate that this T
could be the result of a deamination of a methylated C,
which may occur asymmetrically, as we have only
observed this occurrence in the T-C-T allele and not
the C-C-C allele. Embryonic stem cells are known to
possess non-CpG methylation at CpA and CpT sites,
which appears to be mediated in part by Dnmt3a
(Ramsahoye et al., 2000). Additionally, homologous
recombination of direct repeats of GFP facilitated by I-
SceI in mouse ESCs has been found to stimulate de
novo methylation through Dnmt1 that leads to
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13
silencing of the recombined cassette (Cuozzo,
Porcellini et al., 2007). These findings combined with
the consistent presence of the mutation when using
destabilized Cas9-DD lead us to speculate that DSBs
induced by destabilized Cas9-DD could be repaired in
a different manner than those of WT-Cas9 and
stabilized Cas9-DD. Additionally, the lack of knock-in
events when using destabilized DD-Cas9 suggests that
DD-Cas9 may be preferable for restricting Cas9
activity. This observation is in accordance with the
initial work of Banaszynski et al. (2006) describing N-
terminal DD fusions as inherently less stable than C-
terminal fusions.
Analyzing cells in bulk is informative, but it
lacks the resolution only possible at the single-cell (or
–clone) level. When working with hiPSCs, single-cell
analysis is possible, albeit rather difficult, as the
current method for obtaining pure clones is to single-
cell sort cells into numerous 96-well plates and analyze
the survivors once colonies have formed. The use of a
sib-selection-based strategy for enrichment of
modified cells is useful, but this strategy is dependent
on subsequent subcloning for isolating a pure
population as well (Miyaoka et al., 2014). Thus, in our
analyses, we investigated the consequences of KiBL via
single-cell PCR. Cell I was found to have only received
the 5’sgRNA vector, but the cassette was knocked-in
precisely without loss of any nucleotides, underscoring
that KiBL is compatible with single sgRNAs as well.
Cell II received both sgRNA vectors, and the modified
allele possessed a precise 5’ genomic-imprecise 5’
cassette junction and a 3’ precise cassette-imprecise 3’
genomic junction, which is somewhat consistent with
destabilized Cas9-DD targeting a nearby incorrect
PAM. Interestingly, in both cells, the other allele was
wholly correct except for an additional A at the
cleavage site of the 5’ sgRNA. It is possible that this
additional A is the result of error-prone NHEJ or that
there is a small subpopulation of cells in culture that
acquired an A at this position undergoing KiBL.
Further analysis involving deep sequencing and more
single-cell PCR would have to be carried out to
determine which actually occurred. It is worth noting
that single-cell cloning is the major obstacle to the
application of the CRISPR/Cas9 system to hiPSCs.
Additionally, our analysis suggests that multi-sgRNA
KiBL may benefit from having both sgRNAs present
within the same vector, similar to what has been
developed for multiplexed editing using lentiviral
vectors (Kabadi et al., 2014).
In this work, we chose to identify off-target
KiBL events through a targeted, candidate-based
method utilizing off-target sites possessing the fewest
number of mismatches. While this method provides a
good starting point, it does not necessarily take into
account the finding that SpCas9 can tolerate bulges in
the target DNA and the sgRNA, as well as cleave at
non-canonical PAMs and tolerate more than four
mismatches (Lin et al., 2014; Tsai et al., 2014b).
Whole genome sequencing would identify the off-
target KiBL events, in addition to error-prone off-
target cleavage repair, but its cost makes it prohibitive
for hiPSCs, and the additional information may not be
worth the increased cost, as error-prone off-target
repair appears not to occur at high frequency in
hiPSCs and primary stem cells (Mandal et al., 2014;
Yang, Grishin, Wang et al., 2014). There are currently
three unbiased methods for identifying off-target
cleavage events: GUIDE-seq, Digenome-seq, and
identification through integrase-deficient lentiviral
vectors (Tsai et al., 2014b; Kim et al., 2015; Wang et
al., 2015). GUIDE-seq and identification through
integrase-deficient lentival vectors are functionally
similar, and it does not escape us that KiBL could be
used in a similar approach. Ideally, this would be
combined with a linear amplification-based method to
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14
maximize sensitivity. Such a strategy has been
previously used to examine translocations generated
by TALENs and CRISPR/Cas9 and to measure the
frequency of promoterless gene targeting using adeno-
associated virus vectors (Frock et al., 2014; Barzel et
al., 2015).
While being able to place exogenous sequences
at any genomic location is useful, being able to knock-
in sequences at loci where transgenes do not disrupt
endogenous gene regulation (known as safe harbor
sites) is also useful. We chose the H11 as our target
locus because it has previously been identified as a safe
harbor site in the human and mouse genome (Zhu et
al., 2014; Tasic et al., 2011). The use of a safe harbor
site is of great importance for the placement of
transgenes in cell therapies because of the potential for
the integration event itself to perturb endogenous
genes in a possibly oncogenic manner (for further
information see Sadelain, Papapetrou, and Bushman
et al., 2011). Additionally, the gene order of the H11
locus appears to be highly conserved, particularly with
respect to mammals and through vertebrates in
general. For example, the order appears even to be
conserved in the coelacanth. This conservation makes
H11 particularly attractive as a genome engineering
site.
Ultimately, we have presented a method for
utilizing the CRISPR/Cas9 system to facilitate in vivo
blunt-end cloning in precise, homology-independent
manner through NHEJ. This method, KiBL, is made
possible by the ability of Cas9 to make blunt DSBs,
which we and others have exploited using two sgRNAs
at once to facilitate precise excision of the intervening
sequence (Cong et al., 2013; Mali, Yang et al., 2013;
Canver, Bauer et al., 2014; Zheng, Cai et al., 2014).
The main advantages of our method are its relatively
high frequency of precise genomic-cassette junctions,
its lack of incorporation of additional exogenous
sequences, and its independence from sgRNA
efficiency. Additionally, KiBL may prove amenable to
high-throughput applications, whereas HR is not, due
to the necessity of constructing homology arms for
each targeted locus. This construction is of particular
concern for the concept of saturation editing, which
could become very expensive for targeting large
numbers of loci (Findlay, Boyle et al., 2014). The main
drawbacks of our method are generating large
amounts of cassette (which can be easily overcome)
and the lower electroporation efficiency of linear
dsDNA. The limitation of the amount of cassette
generated greatly affects directly comparing KiBL to
CRISPR/Cas9-mediated HR, because HR uses at least
10-fold more donor vector than our method (≥ 2μg
versus 200ng; Byrne et al., 2014). Thus, there is more
donor vector available for incorporation in HR relative
to KiBL. We recommend that the cassettes be made
with unphosphorylated primers each possessing at
least three phosphorothioate bonds in the 5’ termini.
This property is exemplified in comparing KiBL to the
homology-independent knock-in method used in
zebrafish (Auer et al., 2014). Where we have observed
high levels of precise genome-cassette junctions in our
hiPSCs, Auer and colleagues (2014) observed only 17%
of targeted embryos possessed perfect repair. This
decreased level of precise repair may be due to
unprotected free ends of the cleaved plasmid, as well
as the capability of a perfectly repaired site to be
recleaved by the same sgRNA:Cas9 complex.
Interestingly, our observation of a lack of 0/0 precise
repair when targeting the MYOD1 3’UTR with our
PAMs 3’ sgRNA pair (which regenerates a perfect
target site for one of the sgRNAs) lends additional
support to recleaving by Cas9. This finding, combined
with the rest of our analysis of paired sgRNA-mediated
deletions and observations by others (Cong et al.,
2013; Mali, Yang, et al., 2013; Canver, Bauer et al.,
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15
2014; Byrne et al., 2014; Ghezraoui et al., 2014) also
underscores that NHEJ resulting from blunt,
chemically unmodified DSBs, such as those made by
Cas9, are repaired with a high degree of precision, and
that this may be the predominant NHEJ repair
mechanism (reviewed in Bétermier, Bertrand, and
Lopez, 2014). This observation suggests that the
error-prone NHEJ previously observed when only one
sgRNA is used may be the result of an alternative
repair pathway activated by consistent recleavage of
the target site by Cas9. While we have mainly focused
on using paired sgRNAs for KiBL, our own data also
demonstrate that KiBL can be performed with one
sgRNA as well.
KiBL offers a viable alternative to HR and may
be a better choice for targeting some cell types. For
example, aged hematopoietic stem cells and aged
skeletal muscle stem cells retain the capacity to
precisely repair DSBs through NHEJ, making KiBL the
method of choice for genome engineering in them,
particularly in vivo or in their quiescent, i.e., non-
dividing, state (Flach et al., 2014; Beerman et al.,
2014; Vahidi Ferdousi et al., 2014). In summary, KiBL
is a versatile method capable of facilitating advanced
genome engineering strategies and providing new
insights into how Cas9-induced DSBs are repaired.
Materials and Methods
Choice of sgRNAs and Vector Construction. All
sgRNAs were chosen using the MIT CRISPR Design tool
(http://crispr.mit.edu/). Briefly, genomic regions consisting
of up to 250 bp were chosen for each locus, and the highest
quality guides were chosen for cloning into the pX330
(Addgene #42230) backbone following the protocol
developed by Feng Zhang’s lab
(https://www.addgene.org/static/cms/filer_public/95/12/9
51238bb-870a-42da-b2e8-
5e38b37d4fe1/zhang_lab_grna_cloning_protocol.pdf).
These regions appear in Table 1. All oligos, primers, and
gBlocks were ordered from Integrated DNA Technologies
(IDT, Coralville, IA). A description of the sgRNAs chosen, as
well as the oligos used for cloning, appear in Table 2.
sgRNA/Cas9 vectors, as well as all additional plasmids in
this study, were isolated using the Nucleobond Midi Plus EF
kit (Machrey-Nagel, Bethlehem, PA).
The C-terminal L106P destabilization domain was
obtained as a gBlock (IDT), and was digested with FseI (New
England Biolabs (NEB), Ipswich, MA) and BsaI-HF (NEB).
pX330 was digested with FseI and EcoRI-HF (NEB), and
dephosphorylated with Antarctic phosphatase (NEB). The
digested C-terminal DD and the pX330 backbone were
ligated with T4 DNA ligase (NEB), and transformed into
STBL3 chemicompetent cells (Life Technologies, Grand
Island, NY). In order to clone sgRNAs into pX330-Cas9-DD,
the BbsI cloning site was replaced with a BsaI x2 cloning site
using a gBlock containing the entire sgRNA expression
cassette. Both pX330-Cas9-DD and the gBlock were
sequentially digested with PciI (NEB) and KpnI-HF (NEB)
before dephosphorylation of the backbone and subsequent
ligation and transformation to generate pX330-BsaIx2-
Cas9-DD. This new cloning site uses FastDigest Eco31I
(isochizomer of BsaI; Thermo Fisher Scientific, Waltham,
MA) and the two following general primers:
Sense oligo: 5’- accg-NNNNNNNNNNNNNNNNNNNN-3’
Antisense oligo: 5'-aaac-NNNNNNNNNNNNNNNNNNNN-
3'
Additionally, pX330-Cas9-DD versions of the H11 sgRNAs
were generated by cloning Cas9-DD directly into their pX330
vectors through the use of FastDigest BshTI (Thermo Fisher
Scientific) and FastDigest NotI (Thermo Fisher Scientific).
To generate pX330-BsaIx2-DD-Cas9, the N-
terminal DD was obtained as a gBlock and digested with
FastDigest BshTI and FastDigest BglII (Thermo Fisher
Scientific) before being cloned into the pX330 backbone that
had been digested with the same enzymes and
dephosphorylated with FastAP (Thermo Fisher Scientific).
This vector was subsequently digested with FastDigest KpnI
(Thermo Fisher Scientific) and FastDigest NotI to isolate the
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16
DD-Cas9 insert. pX330-BsaIx2-Cas9-DD was digested with
the same enzymes and dephosphorylated to remove Cas9-
DD. The DD-Cas9 insert was cloned into the pX330-BsaIx2
backbone to generate pX330-BsaIx2-DD-Cas9.
Knock-In Cassette Construction. The DICE-EPv2.0 and
PTKmChR cassettes were amplified from a common vector
containing the PuroRΔTK fusion gene (from Addgene #
22733) followed by a P2A ribosomal skipping element and
mCherry followed by the rabbit beta-globin terminator. This
cassette is under the control of the mouse phosphoglycerol
kinase (PGK) promoter. Cassettes were amplified via PCR
with Q5 high-fidelity polymerase, subjected to gel
electrophoresis, excised, and purified with the MinElute Gel
Extraction Kit. For the DICE-EPv2.0 cassettes, the phiC31
and Bxb1 attP sites were included in the primers used for
amplification. All primers used for cassette amplification
appear in Table 3.
The pKER cassettes were assembled by first
obtaining phosphorylated gBlocks for Clover, mRuby2, and
mCerulean in the form of EF1α promoter-fluorescent
protein-rabbit beta globin terminator. These three gBlocks
were cloned separately into a kanamycin resistance
backbone with blunt ends. mCardinal and mKOκ were
obtained as gBlocks, digested with FastDigest NheI (Thermo
Fisher Scientific) and FastDigest NotI, and cloned into a
dephoshorylated pKER-Clover backbone that had been
digested with the same enzymes to remove Clover. Cassettes
were amplified from 1-3 ng of plasmid using Q5 high fidelity
polymerase in eight individual reactions. After
amplification, the reactions were pooled and purified using
either the MinElute PCR Purification kit (Qiagen) or the
GeneJet PCR Purification kit (Thermo Fisher Scientific).
The purified reaction was then treated with FastDigest DpnI
to digest residual plasmid. After digestion, the reaction was
brought to 100 µL with nuclease-free water (Life
Technologies) and purified with a CHROMASpin-1000+TE
chromatography column (Clontech, Mountain View, CA) to
remove digested plasmid and restriction enzyme. Barcoded
cassettes containing homology arms were first amplified with
primers containing, in the 5’direction, a 20-base secondary
priming sequence, a 6-base randomized barcode, and the
priming sequence for the pKER cassette. This reaction was
then purified with the GeneJet PCR Purification kit and used
as the template for secondary PCR using primers containing
50-base homology arms and the secondary priming
sequence, which was purified and digested in the same
manner as described above. All primers used for cassette
amplification appear in Table 3.
Cell Culture. HEK293 cells were maintained in 6-well
tissue culture plates coated with Poly-L-Lysine (Sigma-
Aldrich, St. Louis, MO) in high-glucose DMEM (Thermo
Fisher Scientific) supplemented with 10% fetal bovine serum
(FBS; Gemini Bio-Products, West Sacramento, CA), 1x non-
essential amino acids (Life Technologies), and 1x GlutaMAX
(Life Technologies). Cells were passaged with 0.05%
Trypsin-EDTA (Life Technologies) as needed at a 1:12 split.
C2C12 cells were maintained in the same media as HEK293
cells and passaged in the same manner. C2C12 cells were
grown on 6-well tissue culture plates. H9 hESCs were
maintained in 0.1% gelatin-coated 6-well tissue culture
plates on γ-irradiated mouse embryonic fibroblast feeder
cells in human embryonic stem cell media consisting of
DMEM/F12 (Thermo Fisher Scientific), 20% Knockout
Serum Replacement (Life Technologies), 1x non-essential
amino acids (Life Technologies), 1x GlutaMAX (Life
Technologies), 0.1mM β-mercaptoethanol (Life
Technologies), and 8ng/mL bFGF (PeproTech, Rocky Hill,
NJ). Cells were passaged as clumps using collagenase IV
(Stem Cell Technologies, Vancouver, British Columbia,
Canada). JF10 hiPSCs were maintained in 6-well tissue
culture plates on recombinant human vitronectin (Life
Technologies) in Essential 8 media (Life Technologies).
Cells were passaged as clumps every three to four days with
Versene (Life Technologies) at a 1:6 split.
Detection of CRISPR/Cas9-Facilitated Excision.
HEK293 and C2C12 cells were transfected using FuGene HD
(Promega, Madison, WI). Briefly, 150,000 cells were plated
one to two days prior on poly-L-lysine-coated (HEK293) or
standard (C2C12) 24-well tissue culture plates and were
transfected with 5 μg (HEK293) or 3 μg (C2C12) of each
sgRNA/Cas9 vector for a total of 10 μg or 6 μg per
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17
transfection, respectively, in Opti-MEM I (Life Technologies)
media with FuGene HD being used at a 3:1 ratio. Cells were
harvested four days post-tranfection and genomic DNA was
isolated using the DNeasy Blood and Tissue Mini Kit
(Qiagen). Each transfection was separately performed at
least twice. H9 hESCs were transfected using the Amaxa
Nucleofector (Lonza, Basel, Switzerland) with the Human
Embryonic Stem Cell Nucleofection Kit 2 (Lonza). Briefly,
cells were passaged normally into Matrigel-coated 6-well
plates three days prior to nucleofection. Before
nucleofection, media was replaced at least 1 hr beforehand
with fresh media containing 10μM ROCK inhibitor Y-27632
(R&D Systems, Minneapolis, MN). Cells were dissociated
with collagenase IV, and subsequently electroporated with
1.5 μg of each sgRNA/vector for a total of 3 μg using program
B-16 according to the manufacturer’s instructions, and
plated on 0.1% gelatin-coated 12-well plates containing at
one reaction per well in human stem cell media containing
10μM ROCK inhibitor Y-27632. Each reaction was carried
out at least twice Genomic DNA was isolated from the cells 2
days later with the DNeasy Blood and Tissue Mini Kit.
PCR amplification of the deletion-containing
regions was carried out using OneTaq (NEB) and Q5 high
fidelity polymerase using 150 ng of gDNA according to the
manufacturer’s instructions. Amplicons were subjected
agarose gel electrophoresis, and subsequently the deletion-
containing bands, as well as the full-length bands where
applicable, were excised and purified using the MinElute Gel
Extraction Kit (Qiagen). Purified amplicons were then
subcloned using the CloneJet PCR Cloning Kit (Thermo-
Fisher Scientific), and subsequently transformed into α-
Select electrocompetent cells (Bioline, Taunton, MA), 10-β
chemicompetent cells (NEB), or STBL3 chemicompetent
cells according to their manufacturers’ instructions.
Colonies were inoculated and grown overnight. Plasmids
were isolated the following day using the Miniprep Spin Kit
(Qiagen), and subsequently subjected to Sanger sequencing
of the inserts. Primers used for amplification of excision
events appear in Table 4.
Flow Cytometry. Flow cytometric analysis was carried out
on LSRII- and FACScan-class analyzers (BD Biosciences, San
Jose, CA). Sorting was carried out on FACSAriaII-class
sorters (BD Biosciences). Live cells were discriminated on
the basis of DAPI exclusion using the NucBlue Fixed Cell
Stain ReadyProbes reagent (Life Technologies). mCherry
was detected via excitement with a 561 nm yellow/green 100
mW laser with a 571 nm LP filter in the optical path, a 725
nm SP splitter, and a 690±40 nm BP filter. Clover was
detected via excitement with a 488 nm 50mW blue laser, a
505 nm LP splitter, and 525±50 nm BP filter. mKOκ was
detected via excitement with a 532 nm 150mW green laser
and a 575±25 nm BP filter. mCardinal was detected with a
640 nm 40mW red laser and a 670±30 nm BP filter.
mRuby2 was detected with a 532 nm 150mW green laser, a
600 nm LP splitter, and a 610±20 nm BP filter. mCerulean
was detected with a 405 nm 50mW violet laser and a 450±50
nm BP filter in the absence of DAPI. All flow cytometric data
were analyzed using FlowJo software (Tree Star, Ashland,
OR).
Detection of Knock-in Blunt Ligation and
Homologous Recombination. For HEK293 cells,
FuGene HD was used to transfect cells as described above,
using 100 ng of cassette and 1.5 µg of each sgRNA/Cas9
vector for a total of 3.1 µg of DNA. 500,000 cells had been
plated one to two days prior to transfection. Reactions were
carried out in triplicate. Shield-1 (Clontech) was added to
the cells immediately prior to transfection at a concentration
of 1 µM. Puromycin (Life Technologies) selection was
started two days after tranfection at a concentration of
1µg/mL and carried out for four days with fresh media and
antibiotic being replaced every two days. Cells were
analyzed by flow cytometry two days after transfection or six
days in the case of puromycin-selected cells. Briefly, cells
were trypsinzed with 0.05% trypsin, resuspended in PBS
(Thermo Fisher Scientific) with 2% FBS, and were filtered
before analysis to remove clumps and debris. gDNA was
isolated from the remaining cells with the DNeasy Blood and
Tissue Mini Kit. Genome-cassette junctions were amplified
with Q5 Hi-Fidelity polymerase using at least 100 ng of DNA
per reaction. Amplicons were subjected to gel
electrophoresis, excised, purified, subcloned, transformed,
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18
and sequenced as described above. Primers used appear in
Table 5.
JF10 cells were plated three to four days prior to
electroporation on vitronectin-coated 6-well plates. Cells
were incubated at least one hour beforehand with Essential 8
media containing 10 µM ROCK inhibitor Y-27632. Cells
were then dissociated into single cells with Versene. Cells
were resuspended in the homemade nucleofector buffer
described in Zhang, Vanoli et al., 2014, with 500ng of each
sgRNA/Cas9 vector and 200ng of cassette per reaction (for a
total of 1.2 µg of DNA) and electroporated with the Amaxa
nucleofector using program B-16 in triplicate.
Electroporated cells were plated on vitronectin-coated 12-
well plates in Essential 8 media containing 10 µM ROCK
inhibitor Y-27632 and 0, 0.5, or 1 µM of Shield-1. ROCK
inhibitor was added each day for four days after
nucleofection. Modified cells were subjected to FACS four to
seven days after nucleofection. Briefly, cells were treated 1
hour prior with 10 µM ROCK inhibitor Y-27632 before being
trypsinized with TrypLE (Life Technologies) for 10 minutes.
Harvested cells were resuspended in DPBS (Life
Technologies) with 2% AlbuMAX I (Life Technologies), 2
mM EDTA (Life Technologies), NucBlue Fixed Cell Stain
ReadyProbes reagent, and 10 µM ROCK inhibitor Y-27632,
and subsequently filtered to remove clumps and debris.
Genomic DNA was isolated from sorted cells using the
ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA).
gDNA was then subjected to targeted amplification of the
H11 locus via PCR amplification with Q5 high-fidelity
polymerase or multiple displacement amplification (MDA; a
variant of whole genome amplification) following Dean et al.,
2002, with the addition of inorganic (yeast) pyrophosphatase
(NEB). PCR-amplified products were purified with the
MinElute PCR Purification kit (Qiagen), diluted to 100 µL
with Buffer EB (Qiagen), and used for further genome-
cassette junction PCR amplifications. MDA reactions were
diluted to 200 µL with TE-EF buffer (Machrey-Nagel) and
also used for further genome-cassette junction PCR
amplifications. Primers used appear in Table 5.
Analysis of Off-Target KiBL Events. For each sgRNA,
forward and reverse primers were designed to each of the top
six off-target sites as predicted by the MIT CRISPR Design
Tool. Each forward and reverse primer was used in
conjunction with the 5’ and 3’ pKER-Clover detection
primers, for a total of 48 reactions per sample. 1 μL of 1:200
diluted WGA of Clover+ cells was used in each PCR
amplification. PCR was carried out with Q5 high-fidelity
polymerase. Off-target primers appear in Table 6.
Western Blotting. HEK293 cells in 6-well plates that had
been plated two days prior were transfected using FuGene
HD with 5 µg of plasmid encoding the H11-r1-2 sgRNA and
WT-Cas9, Cas9-DD, or DD-Cas9 as described above. Cas9-
DD and DD-Cas9 were transfected in duplicate. All wells
received 0.5 µM Shield-1 except one replicate each for Cas9-
DD and DD-Cas9. The following day, media was aspirated
and replaced with fresh media containing 0.5 µM Shield-1
for all wells except for the replicates that did not receive
Shield-1 the previous day. 5 hours later, media was
aspirated, cells were washed twice with cold PBS, and
protein was extracted with RIPA buffer (Thermo Fisher
Scientific) containing 1x HALT protease inhibitor cocktail
(Thermo Fisher Scientific). Cell lysate was snap-frozen for
later analysis. Protein concentration was determined with
the Bradford assay.
For Western blotting, 50µg of protein was
denatured in the presence of 1x Laemmli buffer (Bio-Rad
Laboratories, Hercules, CA) and 0.1 M DTT (Life
Technologies) at 100°C for 5 minutes. Samples were then
resolved on a 10% SDS-PAGE gel (Bio-Rad) at 100 V in 1x
Tris/Glycine/SDS buffer (Bio-Rad). Protein was transferred
to an Immuno-Blot PVDF membrane (Bio-Rad) under wet
transfer conditions at 200 mA for 1 hour with constant
current at 4°C in 1x Tris/Glycine/SDS buffer + 10%
methanol. Following transfer, the membrane was blocked
for 1 hour at room temperature with agitation with Protein-
Free T20 blocking buffer (Thermo Fisher Scientific) and
incubated overnight with agitation at 4°C with 1:10,000
monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich) and
1:10,000 monoclonal anti-GAPDH antibody (clone GAPDH-
71.1; Sigma-Aldrich). The following day, the membrane was
washed three times for 5 minutes each with PBS + 0.05%
TWEEN-20 (Sigma-Aldrich). Secondary incubation was
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The copyright holder for this preprint. http://dx.doi.org/10.1101/019570doi: bioRxiv preprint first posted online May. 21, 2015;
19
carried out at room temperature with agitation for 1 hour
with 1:5000 goat anti-mouse IgG conjugated to horseradish
peroxidase (Thermo Fisher Scientific). The membrane was
washed again as described above and then incubated for 5
minutes at room temperature with Clarity Western ECL
substrate (Bio-Rad). Amersham Hyperfilm ECL (GE
Healthcare, Buckhamshire, UK) was exposed to the
membrane and subsequently developed. The blot was then
quantified using ImageJ and normalized to the WT-Cas9
sample.
Statistics. All statistical analysis was carried out using
GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).
Acknowledgments
We thank Jennifer Sullivan for designing the off-target
primers. We thank Chris Bjornson for comments on
and critical reading of the manuscript. We thank
Michael Wilkinson for comments and discussion. We
also thank the Jain Foundation and Cellular Dynamics
Inc. for providing us with the JF10 hiPSCs.
Additionally, we thank the staff of the Stanford Shared
FACS Facility, particularly Cathy Crumpton and
Ometa Herman for sharing their expertise.
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Figure 1. Analysis of deletion repair generated by paired sgRNAs. (A) Schematic showing excision of sequence between two sgRNAs followed by how deletion repair was analyzed. (B) Each paired PAM orientation has four possible outcomes for precise repair (depicted below each orientation) based on SpCas9’s ability to cleave at the third or the fourth base upstream of the PAM. (C-F) (i) Schematic diagrams of the targeted locus with sgRNA targets depicted as sgRNA/Cas9 complexes. Additionally, the largest deletion size for each locus is depicted. Red indicates exonic sequence and purple indicates 3’UTR. (ii) Graphs depicting the cumulative percentage of precise (green), excision+insertion (orange), and imprecise (red) repair observed in sequenced amplicons for each tested sgRNA pair. (iii) Dot plots of deletion-containing amplicons described in sub-panel ii. Each dot represents the sum of bases beyond 0/0 precise repair. The numbers of amplicons analyzed for each sgRNA pair appear on the subpanel iii graphs. Data from panel iii graphs were analyzed with the Kruskal-Wallis test followed by post-hoc Dunn’s multiple comparisons test. For MYOD1, PAX3, and mUtrn, all sgRNA pairs were compared against all pairs. For PAX7, each pair was only compared between cell types. **** = P < 0.0001; ** = P < 0.004.
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Figure 2. Knock-in blunt ligation (KiBL) in human immortalized cells. (A) The use of two sgRNAs at the same locus should facilitate ligation of exogenous sequence in place of the excised sequence. (B) Schematic of the PGK-PTKmChR and DICE-EPv2 cassettes. (C) Workflow for KiBL experiments: HEK293 cells are transfected with sgRNA/Cas9 vectors and cassettes before flow cytometric analysis and sequence analysis. (D) Schematic diagram of the PAX7 3’UTR region along with sgRNAs used in these experiments. (E) Representative flow cytometric data displaying the difference in the percentage of mCherry+ cells between the DICE-EPv2.0 cassette transfected alone and with sgRNA/Cas9 vectors after two days. (F) Quantification of the DICE-EPv2 transfection experiments in terms of percentage of mCherry+ cells using sgRNAs PAX7r2-1 and PAX7r3-2. n = at least 2 independent experiments consisting of 3 replicates each for each transfection Data is shown as the mean ± SEM. (G) Quantification of the PGK-PTKmChR cassette transfections. Same experimental conditions as in panel F. n = at least 2 independent experiments consisting of 3 replicates each for each transfection. Data is shown as the mean ± SEM. (H) (i) Graph of cumulative percentage of sequenced amplicons categorized by repair status of the genome-cassette junction. n for each is displayed on the graph. (ii) An example chromatogram of the precise genomic/precise cassette junction for the PTKmChR cassette. Boxed sequence denotes PGK promoter sequence, PAM is underlined, and the first four bases of sgRNA target are overlined.
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Figure 3. pKER cassettes are suitable for use in knock-in blunt ligation. (A) Schematic diagrams of the pKER series of cassettes organized by brightness. Each cassette consists of a fluorescent protein under the control of the EF1α promoter and followed by the rabbit beta-globin terminator sequence. (B) Schematic diagram of the 5’ side of the H11 locus on human chromosome 22 depicting the four sgRNAs positioned relative to the sense strand. 548 bp separates the 5’-most and the 3’-most sgRNAs from each other. (C) Diagram depicting the workflow for analyzing pKER expression. pKER cassettes and pairs of sgRNA/Cas9 vectors are co-transfected into HEK293 cells and analyzed by flow cytometry after two days. (D) Representative flow cytometric data generated with pKER-mKOκ and the H11 sgRNAs. (E) Quantification of the percentage of mKOκ+ cells and their relative median fluorescence intensity for phosphorylated and unphosphorylated cassettes for all four possible PAM orientations. n = 3 independent experiments each consisting of 3 technical replicates. Data is displayed as the mean ± SEM of the averages of each experiment and were analyzed using a two-way ANOVA with repeated measures followed by a post hoc Tukey’s multiple comparisons test. ** = P < 0.003.
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Figure 4. Cas9-DD and nuclease protection facilitate a higher degree of precise genome-cassette junction repair. (A) Diagram of Cas9-DD showing the location of the destabilization domain on SpCas9. The domain is near the PAM-binding domain at the C-terminus. (B) Schematic diagram displaying the workflow for comparing WT-Cas9 to Cas9-DD. HEK293 cells are transfected with pKER-Clover cassette and the PAX7 PAMs Out-Large pair of vectors encoding either WT-Cas9 or Cas9-DD. Two days post-transfection, a portion of the cells are subjected to flow cytometric analysis and the remainder are reserved for genomic DNA isolation for sequence analysis. (C) Representative flow cytometric data for the pKER-Clover cassette and the PAX7 PAMs Out-Large sgRNAs with destabilized Cas9-DD. (D) Quantification of the percentage of Clover+ cells and their relative median fluorescence intensity for phosphorylated and unphosphorylated cassettes for WT-Cas9, destabilized Cas9-DD, and stabilized Cas9-DD (1 µM Shield-1 for 24 hours). n = 5 independent experiments each consisting of 3 technical replicates. Data is displayed as the mean ± SEM of the averages of each experiment and were analyzed using a two-way ANOVA with repeated measures followed by a post hoc Tukey’s multiple comparisons test. ** = P < 0.001. (E) Diagram of the modified PAX7 3’UTR locus. This diagram displays pKER-Clover placed at the locus in the 5’ to 3’ orientation. Arrows indicate the primers used to generate amplicons for the sequence analysis in (F). (F) Analysis of genome-cassette junctions for the comparison of WT-Cas9 with destabilized Cas9-DD and the determination of the effect of protecting the cassette from nuclease degradation. Numbers of amplicons analyzed appear beneath their respective treatments. Also depicted are the sequences of the amplicons from the destabilized Cas9-DD junction analysis. Black denotes genomic sequence, turquoise denotes cassette sequence, PAM is underlined, and dashes denote unobserved sequence.
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Figure 5. Stabilized Cas9-DD facilitates knock-in blunt ligation in hiPSCs. (A) Schematic diagram of the targeted H11 locus in JF10 hiPSCs showing the sequence variation at three regions. Individual bases of interest are indicated with an arrow. SNPs are indicated in red. The middle region depicts a homozygous cytosine in violet 9bp upstream of the PAM of the 5’ sgRNA that differs from human reference sequence. (B) Diagram of the workflow for examining the efficiency KiBL in hiPSCs. Cells are electroporated with 200 ng of unphosphorylated phosphorothioate (UPT) pKER-Clover and 500 ng of each vector encoding the H11 PAMs Out sgRNA pair for WT-Cas9 or Cas9-DD. Four to seven days later, Clover+ cells are isolated with FACS for genomic DNA extraction and subsequent downstream analysis of genome cassette junctions. (C) Comparison of WT-Cas9- and Cas9-DD-mediated KiBL at the H11 locus via fluorescence microscopy. Scale bar = 50 µm. (D) Examples of FACS plots from KiBL using UPT-Clover. Plots are of singlet live cells. (E) Quantification of the percentage of Clover+ cells via FACS for KiBL using UPT-Clover and Cas9-DD. n for each condition = at least 2 independent experiments with at least 2 technical replicates each. Each data point represents the mean percentage of Clover+ cells for one experiment. The mean of the experiments is graphed as a horizontal line; the error bars are ± SEM. Data were analyzed using an ordinary one-way ANOVA with a post-hoc Tukey’s multiple comparisons test. * = P < 0.02. (F) Schematic diagram showing targeted amplification strategy of knock-in blunt ligation events at the H11 locus. Full arrowheads denote the primers used to amplify across the locus in the primary PCR. Half arrowheads denote the primers used in conjunction with the full arrowheads to amplify the genome-cassette junctions via nested PCR. (G) Sequence analysis of the 5’-genome 5’-cassette junctions of the KiBL event depicted in panel (F) for various degrees of stability of Cas9-DD. 5’ allele-specific SNP is denoted in red. The 5’ side of the pKER cassette is denoted in turquoise. Nucleotide in green corresponds to Cas9 cleaving at the fourth base upstream of the PAM (underlined) instead of the third. The cytosine colored violet indicates the reference base for JF10 at that position; the thymine in orange indicates a mutation not present in JF10. Dashes indicate unobserved bases.
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Figure 6. Clonality analysis of KiBL using hiPSCs at the H11 locus. (A) Single-cell PCR-based analysis of two cells from independent transfections. Red denotes allele-specific SNPs and de novo mutations, turquoise denotes cassette sequence, black denotes genomic, green denotes wobble-cleaved bases, violet denotes the wild type SNP for JF10 that differs from reference, dashes denote unobserved sequence, and orange denotes an insertion. (B) Representative FACS plot of singlet live hiPSCs transfected with equal amounts (100 ng each) UPT-Clover and UPT-mKOκ cassettes with stabilized Cas9-DD the H11 PAMs Out sgRNA pair. Data is representative of two independent experiments. (C) FACS plot of singlet live hiPSCs transfected with equal amounts (66.67 ng each) UPT-Clover, -mKOκ, and mCardinal cassettes with stabilized Cas9-DD the H11 PAMs Out sgRNA pair. (D) Sequence analysis KiBL events at 5’ H11- 5’ pKER junction resulting from the use of stabilized Cas9-DD and barcoded UPT-Clover cassettes possessing short 50 bp homology arms. Red denotes allele-specifc SNPs (also indicated by an arrow), violet denotes HR-mediated conversion of a base (also indicated by an arrow), blue denotes a base HR-mediated insertion, orange denotes a common index sequence, green denotes the barcode sequence, turquoise denotes the 5’ cassette sequence, dashes indicated unobserved sequence. PAM is underlined.
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Figure 7. Targeted analysis of off-target KiBL events in hiPSCs. Genomic DNA from various CRISPR/Cas9-treated UPT-Clover+ cells was subjected to whole genome amplification and used for analysis of the top six off-target sites (denoted OT1-6) for both the sgRNAs of the H11 PAMs Out pair. Each off-target site was analyzed using a sense (denoted F) and an antisense (denoted R) primer designed to amplify the off-target locus. Each F and R primer were used in two amplifications, one with the 5’ pKER cassette detection primer and the other with the 3’ pKER cassette detection primer. H11 F and H11 R correspond to the primers H11-X1-Fwd and H11-X1-Rev respectively and act as on-target controls. White indicates lack of detection, grey indicates faint detection, and black indicates strong detection. Each Cas9 treatment indicates an independent pool of cells.
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Table 1. Interrogated regions used for design of sgRNAs. The GRCh37/hg19 assembly was used for human regions and the GRC38/mm10 assembly was used for murine regions. Gene Region # Chromosomal Location
MYOD1 1 Hs chr11: 17742664-913
MYOD1 2 Hs chr11: 17743679-928
PAX3 1 Hs chr2: 223066487-736
PAX7 1 Hs chr1: 19062359-608
PAX7 2 Hs chr1: 19062633-845
PAX7 3 Hs chr1: 19061876-2125
mUtrn 2 Mm chr10: 12381712-961
mUtrn 4 Mm chr10: 12384485-734
H11 locus 1 Hs chr22: 31830203-452
H11 locus 2 Hs chr22: 31830847-1096
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Table 2. sgRNAs and cloning oligos used. All sequences are in the 5’ to 3’ direction and relative to the sense strand where appropriate. Dashes separate PAM from target. Lower case indicates overhang sequence for cloning. sgRNA Name Sequence Sense oligo Antisense oligo
MYOD-r1-1 CCT-GCTTACTAACCGAGCCCTCC caccgGGAGGGCTCGGTTAGTAAGC aaacGCTTACTAACCGAGCCCTCCc
MYOD-r1-2 GTCTCAAAGTACTGGGCCCG-GGG accgGTCTCAAAGTACTGGGCCCG aaacCGGGCCCAGTACTTTGAGACc
MYOD-r2-1 TACATTACTACACACGTCCG-TGG caccgTACATTACTACACACGTCCG aaacCGGACGTGTGTAGTAATGTAc
MYOD-r2-2 CCC-TCAGCGACGCCTGTAGGCGG caccgCCGCCTACAGGCGTCGCTGA aaacTCAGCGACGCCTGTAGGCGGc
PAX3-1 CCA-TATTGGTAGCCTGTGACAGG caccgCCTGTCACAGGCTACCAATA aaacTATTGGTAGCCTGTGACAGGc
PAX3-2 AAAATTGATACCGGCATGTG-TGG caccgAAAATTGATACCGGCATGTG aaacCACATGCCGGTATCAATTTTc
PAX3-3 CCT-GTGACAGGGTCCATACTGTA caccgTACAGTATGGACCCTGTCAC aaacGTGACAGGGTCCATACTGTAc
PAX3-4 TTACACAAGGAAGCCCCTGC-TGG caccgTTACACAAGGAAGCCCCTGC aaacGCAGGGGCTTCCTTGTGTAAc
PAX7r1-1 AAGCCTCTGCTTCCGTACTA-TGG caccgAAGCCTCTGCTTCCGTACTA aaacTAGTACGGAAGCAGAGGCTTc
PAX7r1-2 CCT-GGGCGTCCCCCGTCCCCATT caccgAATGGGGACGGGGGACGCCC aaacGGGCGTCCCCCGTCCCCATTc
PAX7r1-3 CCT-CTGCTTCCGTACTATGGCTC caccgGAGCCATAGTACGGAAGCAG aaacCTGCTTCCGTACTATGGCTCc
PAX7r1-4 CCT-GCTTGTTTATGGAGAGCTAC caccgGTAGCTCTCCATAAACAAGC aaacGCTTGTTTATGGAGAGCTACc
PAX7r2-1 CAAACGAATCACTTGAGCCC-AGG caccgCAAACGAATCACTTGAGCCC aaacGGGCTCAAGTGATTCGTTTGc
PAX7r3-2 CCC-ATGCATGAGGGCACGCAAAT caccgATTTGCGTGCCCTCATGCAT aaacATGCATGAGGGCACGCAAATc
mUtrn-r2-1 CCC-AATGGGCTTTAGGAATGTAC caccgGTACATTCCTAAAGCCCATT aaacAATGGGCTTTAGGAATGTACc
mUtrn-r2-3 ATGAAGGCCCAATGGGCTTT-AGG caccgATGAAGGCCCAATGGGCTTT aaacAAAGCCCATTGGGCCTTCATc
mUtrn-r4-1 CCA-ATGGGTCTAAATACGGGCTA caccgTAGCCCGTATTTAGACCCAT aaacATGGGTCTAAATACGGGCTAc
mUtrn-r4-3 ATGCCAATGGGTCTAAATAC-GGG caccgATGCCAATGGGTCTAAATAC aaacGTATTTAGACCCATTGGCATc
H11-r1-1 TTAATTGATCACTCCCCGCA-AGG caccgTTAATTGATCACTCCCCGCA aaacTGCGGGGAGTGATCAATTAAc
H11-r1-2 CCC-CGCAAGGCTAACCTATTTTG caccgCAAAATAGGTTAGCCTTGCG aaacCGCAAGGCTAACCTATTTTGc
H11-r2-1 CCT-CCCGGGTTCAAGCGATTCTC caccgGAGAATCGCTTGAACCCGGG aaacCCCGGGTTCAAGCGATTCTCc
H11-r2-3 GTGTCAGCCTCCTGATTAGC-TGG caccgGTGTCAGCCTCCTGATTAGC aaacGCTAATCAGGAGGCTGACACc
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Table 3. Primers used in cassette amplification. All oligos are in the 5’ to 3’ direction. Primer Name Sequence
DICE-EP-v2-PCR-fwd /5phos/-GAAGAA-
CTCGAGGTAGTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGCGTA-
GCTAGCTACCGGGTAGGGGAGGCGC
DICE-EP-v2-PCR-rev /5phos/-GAAGAA-
GGTACCGGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTGACCAGACAAACCA-
CGCGCCGAGAAGAGGGACAGCTATG
PTKPmChR-PCR-fwd /5phos/-TACCGGGTAGGGGAGGCGCTTTT
PTKPmChR-PCR-rev /5phos/-GAGAAGAGGGACAGCTATGACTGGGAGTAGT
phospho-pKER-fwd /5phos/-GATCTGCGATCGCTCCGGTg
phospho-pKER-rev /5phos/-GAGAAGAGGGACAGCTATGACTGGG
pKER-fwd gatctgcgatcgctccggtg
pKER-rev GAGAAGAGGGACAGCTATGACTGGG
UPT-pKER-fwd G*A*T*CTGCGATCGCTCCGGTG
UPT-pKER-rev G*A*G*AAGAGGGACAGCTATGACTGGG
BarUniv-pKER-fwd AATGATACGGCGACCGATGT-NNNNNN-GATCTGCGATCGCTCCGGTG
BarUniv-pKER-rev CTGCCGACGAGTGTAGATCT-NNNNNN-GAGAAGAGGGACAGCTATGACTGGG
UPT-H11PO-BarCommon-fwd G*A*T*ACTGGAGAGAGGAAGGACTTTATGTAAGTTAATTGATCACTCCCCGC-
AATGATACGGCGACCGATGT
UPT-H11PO-BarCommon-rev A*A*A*AATACAAAATTAGCGGGGCATGGTGGTACCTGCCTGTAATCCCAGCT-
CTGCCGACGAGTGTAGATCT
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Table 4. Excision detection primers. All oligos are in the 5’ to 3’ direction. Primer Name Sequence
MYOD1-X1-fwd CTTTTGGATTGTCCCGGACTCTA
MYOD1-X1-rev CACATCCTCTATACCCAACGAGG
PAX3-X1-fwd GAAAGGCACCTGTAAGGAAACAC
PAX3-X1-rev TGCAATCTGGATCTCTCTGCATT
PAX7-X1-fwd AGGGCACGCAAATCAGGTAA
PAX7-X1-rev AAATGTGGCCACTAGAAATGTTAAG
PAX7-X2-fwd CAAGTGAACATCCCAGTAAGTGAAC
PAX7-X2-rev GAGAGAGTTGGAAACACAGCTTTC
mUtrn-X2-fwd GCACCAATGAACAGGTAATGTAGAG
mUtrn-X2-rev TTTAGCCCAGTAGCTTTCAGTTTTG
H11-X1-fwd CAGAATTTGTTTTGGGATGGGCT
H11-X1-rev TCATGGGGATTCAAGCAGAAAGT
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Table 5. Primers for Amplifying Cassette Junctions. All oligos are in the 5’ to 3’ direction and are to be used in conjunction with the genomic primers in Table 4. Primer Name Sequence
DICE-EP-detection-mch-fwd GACATCCCCGACTACTTGAAGC
PGK-rev1 AAAGAACGGAGCCGGTTGG
pKER-detector-5' TACACGACATCACTTTCCCAGTTTA
pKER-clover-detector-3' CAACCACTACCTGAGCCATCAGTC
pKER-detector-5'-NP TACACGACATCACTTTCCCAGTT*T*A
pKER-clover-detector-3'-NP TAACCACTACCTGAGCCATCAG*T*C
mKOk-AS-N2 CACCTTCAATTGTGAACTCATGCCCATTG
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Table 6. Off-target sites and primers for H11-r1-2 and H11-r2-3. All oligos are in the 5’-3’ direction.
Site Name Off-target Sequence Chromosomal
Location
F Primer R Primer
H11-r1-2-OT1 CAAAACAGGTTAGCCTTGCTAGG
chr1: -45654443
TCACTCAACCAGAATGTTATCCAGT GCTGTAAGGTCCCCTCATTCC
H11-r1-2-OT2 CAAAATGTGGTAGCCTTGCGGGG
chr14: -95753361
GAAAGTGACGGGGACAGTTC CACGGTAGATCAGTCAGGGA
H11-r1-2-OT3 AAATATAGGTTAGCCTTGGGCGG
chr5: -155800441
ACAATTGCTATCCGTTCATTTTCTCTTA TTAAGAGTTGCTCATAATTTAGCTGTCC
H11-r1-2-OT4 GAAACTAGGTTAGCCATGCGTGG
chr19: 1961769
GTGAGGTACTGTCTGGAACTAGG GTTTATTTTTGGATGCGAGTTTTCTCC
H11-r1-2-OT5 CAAAATAGGGTGGCCTTGCAGGG
chr3: -54074337
CGAGAGCTATTTTCTCTAAGGAGGATTA TTCAGTTTGTATTAGAAAACCCTATGCC
H11-r1-2-OT6 TCAAATAGGATAGCCTTGAGTGG
chr10: 75935789
TCATAAAGCATTTACTGATTCCCCC CGGCAGCACGACGTACTAAT
H11-r2-3-OT1 CTCTCAGCTTCCTGATTAGCTGG
chr10: -75885363
TTCTGTCCTTTTTCAGAGTGTGC GAATCTGAAGCTCCCTTTCTCAG
H11-r2-3-OT2 CTGACAGCCTCCTGATTAGGAAG
chr2: 143987130
AGGAGGGCCTCACATATACCTA CAACTAGTGGCTGGAGAGCAA
H11-r2-3-OT3 GTCTGAGCTTCCTGATTAGCAAG
chr7: 18216913
GTTGGGAGAACTGATAATCCGC AGCCTGAAAAGGGTACGGGT
H11-r2-3-OT4 ATGTCAGACTCATGATTAGCGAG
chr13: 68310653
TATGCTCATGCCATTAGGTTCA CCCTTGTGGCAGGATAAGGT
H11-r2-3-OT5 GTGTCAGCCTCCAGATGAGCAGG
chr15: -27274159
CACTTGCTAGCATTCGGCTTC GTAAGATCGCTAGTCAGACCCC
H11-r2-3-OT6 TTGTCAGCCTCTTGATTAGA GAG
chr4: -38229570
ACATGAGAATCCCCAACAAACG TCAGTGGCTCATCAAGTTAGTGT
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Figure 1-figure supplement 1. Sequences of CRISPR/Cas9-mediated deletions at the MYOD1 3’UTR. Underlined blue sequence denotes sgRNA targets. Red denotes PAMs. ~ = Not sequences
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Figure 1-figure supplement 2. Sequences of CRISPR/Cas9-mediated deletions at the PAX3 3’UTR. Underlined blue sequence denotes sgRNA targets. Red denotes PAMs. Dark blue and pink denote overlapping sgRNA target sequence and PAMs respectively.
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Figure 1-figure supplement 3. Sequences of CRISPR/Cas9-mediated deletions at the PAX7 3’UTR in HEK293 cells. Underlined blue sequence denotes sgRNA targets. Red denotes PAMs.
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Figure 1-figure supplement 4. Sequences of CRISPR/Cas9-mediated deletions at the PAX7 3’UTR in H9 hESCs. Underlined blue sequence denotes sgRNA targets. Red denotes PAMs.
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Figure 1-figure supplement 5. Sequences of CRISPR/Cas9-mediated deletions at the mUtrn 3’UTR. Underlined blue sequence denotes sgRNA targets. Red denotes PAMs.
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Figure 2-figure supplement 1. Analysis of PAX7-PTKmChR/DICE-EPv2.0 junctions. PCRs were carried out to analyze the PAX7 5’-5’ cassette and PAX7 3’-5’ cassette junctions in transfected HEK293 cells through agarose gel electrophoresis. The PAX7-X2 primers were used along with the PGK-rev primer for amplification. Marker ladder bands are denoted for reference. Expected band sizes for 5’-5’ junctions: r1-1 + r1-2 = 1188 bp; r1-1 + r1-4 = 1233 bp; r1-2 + r1-3 =1188 bp; r1-3 + r1-4 = 1233 bp; r2-1 = 1595 bp; r3-2 = 808 bp; r2-1 + r3-2 = 808 bp. Expected band sizes for 3’-5’ junctions: r1-1 + r1-2 = 1028 bp; r1-1 + r1-4 = 1028 bp; r1-2 + r1-3 =1036 bp; r1-3 + r1-4 = 1036 bp; r2-1 = 929 bp; r3-2 = 1716 bp; r2-1 + r3-2 = 929 bp.
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Figure 3-figure supplement 1. Stucture and conservation of the H11 safe harbor locus. Schematic from the UCSC Genome Browser (http://genome.ucsc.edu; used Feb. 2009 (GRCh37/hg19) assembly) displaying H11 locus in the human genome on chromosome 22. The flanking genes are DRG1 and EIF4ENIF1. AK074476 is a potential non-coding RNA isolated from human lung. Below are depicted alignments of the region for several vertebrates. Note the high degree of sequence conservation among mammals and the degree of the gene structure conservation among vertebrates.
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Figure 4-figure supplement 1. Stability analysis of Cas9-DD and DD-Cas9 in HEK293 cells. (A) Structure of Cas9-DD and DD-Cas9. (B) Schematic of DD-Cas9. (C) Western blot of stabilized and destabilized Cas9-DD and DD-Cas9 in HEK293 cells. Cells were transiently transfected with vectors encoding sgRNA H11-r1-2 and wild-type Cas9, Cas9-DD, or DD-Cas9, in the presence or absence of 0.5 µM Shield-1 for 1 day post transfection. All versions of Cas9 were detected with an anti-Flag antibody. GAPDH serves as a loading control. Quantification of protein levels as relative fold change normalized to wild-type Cas9 appears to the right.
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Figure 4-figure supplement 2. Analysis of the effect of phophorothioate bonds on KiBL. PCR was carried out to analyze the PAX7 5’-5’ pKER cassette junction in transfected HEK293 cells through agarose gel electrophoresis. The PAX7-x2-fwd and pKER-detector-5’ primers were used in all reactions. The expected band size is 750 bp. Each lane represents one technical replicate for the given condition from the same experiment.
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Figure 5-figure supplement 1. Further analysis of genome-cassette junctions resulting from KiBL at the H11 locus. (A) Chromatogram of bulk sequenced 3’pKER-Clover cassette-3’genome junctions resulting from the use of stabilized Cas9-DD. Boxed sequence is the 3’ end of the cassette. Overlined sequence is the expected first three bases of the 3’ sgRNA target site. Underlined sequence is the PAM. Arrowhead indicates the 3’ allele-specific SNP at this locus. (B) Sequenced amplicons of the 5’ genome-5’ pKER cassette junction from JF10 cells treated with the H11 PAMs Out sgRNA pair and stabilized DD-Cas9 (0.5 µM Shield-1). Left arrowhead indicates 5’ allele-specific SNP (in red). Right arrowhead indicates cytosine that differs from human reference sequence. PAM is underlined. Green denotes fourth base from the PAM. Turquoise denotes cassette sequence.
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Figure 5-figure supplement 2. Comparison of the effect of destabilization of DD-Cas9 on KiBL. PCR was performed to analyze the H11 5’-5’ pKER cassette junction and the H11 locus of sorted JF10 hiPSCs transfected with pKER-mKOκ cassettes and the H11 PAMs Out sgRNA pair with Cas9-DD or DD-Cas9 in the presence or absence of Shield-1. The primers H11-x1-fwd and mKOk-AS-N2 were used to amplify the H11 5’-5’ cassette junction and the primers H11-X1-fwd and H11-X1-rev were used to amplify the H11 locus. Gel electrophoresis was performed to visualize bands. The expected band size of the H11 5’-5’ mKOκ cassette junction is 898 bp. The expected band size of the unmodified H11 locus is 992 bp and the expected size of the KiBL allele is 2233 bp.
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